Methods of using interleukin-2 mutants with reduced toxicity

ABSTRACT

Interleukin-2 (IL-2) mutants having reduced toxicity, which include full-length IL-2, truncated forms of IL-2 and forms of IL-2 that are linked to another molecule are disclosed herein. Particular substitutions within IL-2, particularly within the permeability enhancing peptide region of IL-2 achieve substantial reduction of vasopermeability activity as compared to a wildtype form of the mutant IL-2 while retaining many of the immune activating properties of IL-2. Invention IL-2 mutants can be used to stimulate the immune system of an animal and may be used in the treatment of various disorders and conditions.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/419,960 filed Apr. 7, 2009, which is a continuation of U.S.application Ser. No. 11/093,073, filed Mar. 29, 2005, which is aDivision of U.S. application Ser. No. 10/218,197, filed Aug. 12, 2002,which is an application claiming the benefit under 35 USC 119(e) U.S.Application 60/312,326, filed Aug. 13, 2001, each of which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the use of interleukin-2 (IL-2) as animmunotherapeutic agent and to IL-2 mutants that exhibit reducedvasopermeability and reduced toxicity compared to native IL-2.

BACKGROUND OF THE INVENTION

Cytokines play a role in the growth and differentiation of all cells inthe body but are especially important to cells of the immune system. Acategory of cytokines are called interleukins, of which 18 have beenidentified thus far. Interleukin-2 (IL-2) is an important cytokine forthe regulation of T-cell function in the immune system. Because of itsimportant involvement in both the cellular and humoral arms of theimmune system, IL-2 has been investigated extensively for a potentialrole in the treatment of disease. Although the primary function of IL-2is to stimulate the growth and proliferation of T lymphocytes, IL-2 isalso known to have diverse stimulatory effects on a variety of immunecells, including natural killer (NK) cells, lymphokine-activated killer(LAK) cells, monocytes, and macrophages. In regulating the immunesystem, IL-2 also may trigger the production of secondary cytokines,such as interferons and TNF-α, to further stimulate an immune response.Interferons, interleukins and TNF-α can be made in mass quantitiesthrough recombinant techniques for therapeutic applications.

IL-2 administration is a therapeutic treatment in cancer and otherdiseases. For example, IL-2 is approved for the treatment of metastaticrenal cell carcinoma and melanoma. In this setting, intravenous IL-2produces a 20% rate of remission. However the efficacy of IL-2 has beenrestricted by the relatively severe toxicities associated withtherapeutic dosages. The native form of IL-2 exhibits toxic side effectsthat may include myocardial infarction, renal failure requiringdialysis, fluid retention, nausea and neuropathy. In addition, IL-2administration is associated with generalized inflammatory changes whichinclude the development of dose limiting capillary leak syndrome. Theshort half-life of i.v. administered IL-2 (about 22 minutes) requiresthe higher dosing that leads to toxicity.

Attempts to reduce the unwanted toxicity associated with the therapeuticuse of IL-2 have focused on increasing the half-life of the molecule.This has been achieved by increasing the molecular size by linking IL-2to another molecule such as a protein or polymer, or by linking IL-2 toa targeting molecule such as an antibody. Attempts to direct IL-2 to thesite of disease by a targeting molecule have been somewhat effective andhave resulted in increased levels of therapeutic efficacy, includingcontrol of malignant effusions, prevention of the growth of establishedtumors, and even a reduction in the size of established tumors. However,such approaches cannot be used in all anatomic locations and are notapplicable to disseminated disease.

IL-2 molecules that have a mutated amino acid sequence throughsubstitution of amino acid residues present in the wildtype IL-2molecule have been reported to have reduced toxicity. However, suchmutants are associated with altered biological function such as reducedbinding affinity to forms of the IL-2 cellular receptor and alteredcytokine functions, including T cell stimulation, LAK or natural killercell activation, or secondary cytokine production. Therefore, thereremains a need in the art for a low toxicity variant of IL-2 to minimizetoxicities associated with treatment.

BRIEF SUMMARY OF THE INVENTION

In accordance with the present invention, novel IL-2 mutants withreduced toxicity as compared to native IL-2 are presented. Such mutantsare characterized by substantially reduced vasopermeability activity andsubstantially similar binding affinity for an IL-2 receptor compared toa wildtype form of the IL-2 mutant. By reducing the vasopermeabilityactivity of the IL-2, the present invention meets the need in the artfor a low toxicity variant of IL-2 that avoids toxic side effects suchas vascular leak syndrome. Thus, in one aspect of the present invention,the IL-2 mutant can be used to stimulate the immune system of an animalto achieve maximal therapeutic benefit with reduced side effects.

Invention IL-2 mutants comprise at least one mutation in thepermeability enhancing peptide region of IL-2. In one embodiment, theIL-2 mutant is derived from human IL-2. In another embodiment, the IL-2mutant comprises one or more non-wildtype amino acid residues located atpositions 22-58 of IL-2. Preferred substitutions include W₃₈, G₃₈, Y₃₈,L₃₉, K₄₂ and Y₅₅. The invention IL-2 mutants may be full length IL-2 orfragments of IL-2 and may be linked to another molecule. The above IL-2mutants also may include select mutations outside the permeabilityenhancing peptide region of IL-2.

Also provided is a method for identifying interleukin-2 (IL-2) mutantswith reduced toxicity, the method comprising assaying IL-2 mutantscomprising a mutation in the permeability enhancing peptide region ofIL-2 for vasopermeability activity and for binding affinity for an IL-2receptor, the mutants with reduced toxicity characterized bysubstantially reduced vasopermeability and similar binding affinity foran IL-2 receptor as compared to a wildtype form of the IL-2 mutant.

Further provided is a method of producing a low toxicity IL-2 in a formsuitable for administration in vivo, the method comprising:

a) obtaining a mutant IL-2 characterized by substantially reducedvasopermeability activity and substantially similar binding affinity foran IL-2 receptor compared to a wildtype form of the IL-2 mutant; and

b) formulating the mutant IL-2 with at least one pharmaceuticallyacceptable carrier, whereby a preparation of low toxicity IL-2 isformulated for administration in vivo.

Still further provided is method for stimulating the immune system of asubject in need thereof, the method comprising administering aneffective amount of an interleukin-2 (IL-2) mutant to the subject, themutant comprising a mutation in the permeability enhancing peptideregion of IL-2, the mutant characterized by substantially reducedvasopermeability activity and substantially similar binding affinity foran IL-2 receptor compared to a wildtype form of the IL-2 mutant. Suchmutants can be used as an immunotherapeutic agent in the treatment ofcancers such as renal cell carcinoma or melanoma, in the treatment ofimmune deficiencies such as from viral infection including infection byan immunodeficiency virus, chemotherapy and/or radiation therapy, or inthe treatment of autoimmune disorders.

BRIEF DESCRIPTION OF THE FIGURES

These and other features, aspect, and advantages of the presentinvention will become better understood with regard to the detaileddescription, claims and figures provided herein.

FIG. 1 is a schematic of the IL-2 molecule demonstrating the location ofthe cytokine (shown as solid; approximately amino acids 40-70, and atapproximately amino acids 90-116) and vasopermeability (shown asstippled; amino acids 22-58) activities.

FIG. 2 is a schematic showing the nucleotide sequence (SEQ ID NO:1) andamino acid sequence (SEQ ID NO:2) of a linker within the borderingsequence of human IgG1 heavy chain and human IL-2 that make up achimeric antibody (chTNT-3 heavy chain)/IL-2 fusion protein).

FIG. 3 shows SDS-PAGE analysis (10% polyacrylamide tris-glycine reducedgel) of chTNT-3 antibody, chTNT-3/native IL-2 fusion protein andchTNT-3/IL-2 mutant fusion proteins. The gel was stained with CoomassieBlue. Samples are as follows: biotinylated chTNT-3 (lane 1),chTNT-3/IL-2 (lane 2), chTNT-3/D20K (lane 3), chTNT-3/R38G (lane 4),chTNT-3/R38W (lane 5), chTNT-3/M39V (lane 6), chTNT-3/M39L (lane 7),chTNT-3/F42K (lane 8), chTNT-3/H55Y (lane 9), and molecular weightmarkers (lane 10).

FIGS. 4A-4C profile secondary cytokine secretion by stimulatedperipheral blood mononuclear cells (PBMC) incubated with chTNT-3antibody, chTNT-3/native IL-2, or chTNT-3/IL-2 mutant fusion proteins inserum free media. Cytokine levels representative for the two PBMC donorswere determined by indirect ELISA of culture media for the clays ofculture indicated. FIG. 4A represents interleukin-1β (IL-1β) production.FIG. 4B represents interferon-γ (IFN-γ) production. FIG. 4C representstumor necrosis factor-α (TNF-α) production.

FIGS. 5A-5C depict lymphokine-activated killer (LAK) cell activitygenerated by activation of PBMC with chTNT-3 antibody alone, recombinanthuman IL-2 alone (rhuIL-2), chTNT-3/native IL-2 fusion protein, orchTNT-3/IL-2 mutant fusion proteins. LAK activity was determined by fourhour cytotoxicity activity against Daudi lymphoma cells. FIG. 5A depictsthe R38 mutants. FIG. 5B depicts the M39 mutants. FIG. 5C depicts theD20, F42, and H55 mutants.

FIGS. 6A-6B show tumor therapy using various antibody-IL-2 fusionconstructs. FIG. 6A shows mice receiving chTNT-3/IL-2 (5-20 μg) ascompared to no treatment. FIG. 6B shows mice receiving chTNT-3/IL-2(5-50 μg) as compared to no treatment.

FIGS. 7A-7B show tumor therapy using various antibody-IL-2 fusionconstructs. FIG. 7A shows mice receiving chTNT-3/R38W protein (5-20 μg)as compared to no treatment. FIG. 7B shows mice receiving chTNT-3/R38Wprotein (20-50 μg) as compared to no treatment.

FIG. 8 shows tumor therapy using chTNT-3/N88R protein (5-50 μg) ascompared to no treatment.

FIG. 9 shows the amino acid sequence of full length native human IL-2(SEQ ID NO:3).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided a method foridentifying IL-2 mutants with reduced toxicity, said method comprisingassaying IL-2 mutants comprising a mutation in the permeabilityenhancing peptide region of IL-2 for vasopermeability activity and forbinding affinity for an IL-2 receptor, said mutants with reducedtoxicity characterized by substantially reduced vasopermeability andsimilar binding affinity for an IL-2 receptor as compared to a wildtypeform of the IL-2 mutant. In one embodiment, the mutation comprises asubstitution in at least one non-wildtype amino acids residue located inthe permeability enhancing peptide region of IL-2.

As shown in FIG. 9, mature, native human IL-2 has a 133 amino acidsequence. As used herein, the permeability enhancing peptide region forhuman IL-2 represents residues 22 to 58 (see U.S. Pat. No. 6,008,319).

Vasopermeability activity as seen in FIG. 1 maps to a region of the IL-2that partly overlaps the amino acids believed to be responsible forIL-2's cytokine activity (residues 40-70 and 90-116) (LeBerthon et al.,Cancer Res. 51:2694, 1991; Cotran et al., J. Immunol. 140:1883, 1988).Mutations in the vasopermeability region of IL-2 that are outside of thecytokine region of IL-2, specifically residues 22-39, are preferred.Other segments of the vasopermeability enhancing peptide region of IL-2that are suitable for mutation as disclosed herein include 33 to 58, 37to 58, or 37 to 72.

A substantial reduction in vasopermeability is achieved when the IL-2mutant induces less than approximately 75% of the vasopermeabilityactivity of a wildtype form of the IL-2 mutant. IL-2 mutants of theinvention may induce less than about 50% and even less than about 25% ofsuch vasopermeability activity.

As used herein, a “wildtype form of the IL-2 mutant” is a form of IL-2that is otherwise the same as the IL-2 mutant except that the wildtypeform has a wildtype IL-2 amino acid at each amino acid position of theIL-2 mutant. For example, if the IL-2 mutant is the full-length IL-2(i.e., IL-2 not fused or conjugated to any other molecule), the wildtypeform of this IL-2 mutant is full length native IL-2. If the IL-2 mutantis a fusion between IL-2 and another polypeptide encoded downstream ofIL-2 (e.g., and antibody chain), the wildtype form of this IL-2 mutantis IL-2 with a wildtype amino acid sequence fused to the same downstreampolypeptide. Furthermore, if the IL-2 mutant is a truncated form of IL-2(the mutated or modified sequence within the non-truncated portion ofIL-2), then the wildtype form of this IL-2 mutant is a similarlytruncated IL-2 that has a wild type sequence.

The ability of an IL-2 mutant to substantially decrease vasopermeabilitycan be examined in a pretreatment vasopermeability animal model. Ingeneral, the IL-2 mutant (or the suitable wildtype form of IL-2 mutant)is administered to a suitable animal and, at a later time, the animal isinjected i.v. with a vascular leak reporter molecule whose disseminationfrom the vasculature reflects the extent of vascular permeability. Thevascular leak reporter molecule is preferably large enough to revealpermeability with the wildtype form of the IL-2 used for pretreatment.An example of a vascular leak reporter molecule can be a serum proteinsuch as albumin or an immunoglobulin. The vascular leak reportermolecule preferably is detectably labeled such as with a radioisotope tofacilitate quantitative determination of the molecule's tissuedistribution. Vascular permeability may be measured for vessels presentin any of a variety of internal body organs such as liver, lung, and thelike, as well as a tumor, including a tumor that is xenografted. Lung isa preferred organ for measuring vaospermeability of full-length IL-2mutants.

The Examples appended herewith provide a suitable vasopermeability assayfor testing IL-2 mutants of the invention, particularly where IL-2 islinked to an antibody polypeptide or antibody molecule. In this model,mice xenografted with LS174T human colon adenocarcinoma cells that forma growing solid tumor are pretreated with the mutant IL-2 fused to theDNA targeting antibody TNT-3 that has targeting activity for human tumorcells. The animals are later administered ¹²⁵I-labeled B72.3 monoclonalantibody (a vascular leak reporter molecule), which recognizes the tumorassociated glycoprotein-72 (TAG72) on the LS174T tumor cells. Followinginjection, the percent of the dose of antibody per gram of tumor isdetermined and compared to pretreatment with native IL-2 fused to thesame antibody. Results are expressed as the percent of tumor uptake ofB72.3 per gram of tumor in native IL-2 versus mutant forms of IL-2 (see,e.g., summary in Table 5). A decrease in general vasopermeabilityindicated by a decrease in the percentage dose per gram tumor uptakesignifies a potential for a reduced toxicity of the IL-2 mutant (suchpotential being fully realized in conjunction with the IL-2 mutant'simmune activating properties).

IL-2 mutants which maintain substantially similar affinity for IL-2receptors as compared to a wildtype form of the IL-2 mutant arepreferred. Substantially similar binding to the IL-2 receptor isachieved when the IL-2 mutant exhibits greater than approximately 75% ofthe affinity of the wildtype form of IL-2 mutant for at least one formof the IL-2 receptor. IL-2 mutants that exhibit no more than about 50%of the receptor binding activity compared to a wildtype form of the IL-2mutant may be useful for particular clinical applications.

The affinity of the mutant IL-2 for various forms of the IL-2 receptor(see Theze et al., Immunol Today, 17:481-486, 1996) can be determined inaccordance with well established methods. Binding affinity for thelow-affinity IL-2 receptor (α; p55) and binding to theintermediate-affinity IL-2 receptor (βγ; p70, p75) can be determined inaccordance with the method set forth in the Examples using MT-1 andYT-2C2 cell lines, respectively. Binding affinity of IL-2 mutants forhigh-affinity IL-2 receptor (αβγ; p55, p70, p75), may be evaluated usingHT-2 cells or other cells known to express this form of the IL-2receptor. Other forms of the receptor such as the αβ, αγ and β also maybe evaluated for affinity to the mutants. Alternatively, affinity can bedetermined using receptor subunits such as may be obtained byrecombinant expression (see e.g., Shanafelt et al., Nature Biotechnology18:1197-1202, 2000). Binding of IL-2 mutants to such receptor subunitsand combinations thereof can be determined by standard instrumentationsuch as a BIAcore instrument (Pharmacia).

The ability of an IL-2 mutant to bind to IL-2 receptors may beindirectly measured by assaying the effects of immune activation thatoccur downstream of receptor binding. Such assays include IL-2 inducedcell proliferation (e.g., proliferation of the IL-2-dependent HT-2murine T cell lymphoma cells), tumor regression, viral inhibition,immunomodulating activity (e.g., secondary cytokine induction, such asIL-1β, IFN-γ, and TNF-α from human PBMC), lymphokine-activatedlymphocyte activity, T cell growth, natural killer cell activity (e.g.,measured against Daudi cells), treatment of infections, and the like. Avariety of methods are well known in the art for determining theseimmunological activities of IL-2. Also, details for many of thesemethods are disclosed in the Examples.

The term “IL-2 mutant” or “mutant IL-2” as used herein is intended toencompass any mutant forms of various forms of the IL-2 moleculeincluding full length IL-2, truncated forms of IL-2 and forms where IL-2is linked to another molecule such as by fusion or chemical conjugation.“Full-length” when used in reference to IL-2 is intended to mean thenatural length IL-2 molecule. For example, full length human IL-2 refersto a molecule that has 133 amino acids (see FIG. 9). These various formsof IL-2 mutants are characterized in having a mutation affecting atleast one amino acid position in the permeability enhancing peptideregion of IL-2. This mutation may involve substitution, deletion,truncation or modification of the wildtype amino acid residue normallylocated at that position. Mutants obtained by amino acid substitutionare preferred. Unless otherwise indicated, an IL-2 mutant may bereferred to herein as an IL-2 mutant peptide sequence, an IL-2 mutantpolypeptide, IL-2 mutant protein or IL-2 mutant analog.

A single IL-2 mutant or a mixture of IL-2 mutants may be assayed asdescribed to identify low toxicity mutants. Such mixtures of mutants mayinclude a library of mutants that may be randomized or partiallyrandomized at one or more amino acid positions. Mutant libraries can beprepared by randomizing nucleotides or codons if recombinant expressionof IL-2 is contemplated or by randomizing amino acids if synthetic IL-2is contemplated. Methods for preparing such mutant libraries are wellknown in the art (see, e.g., Ladner, U.S. Pat. No. 5,837,500; Shatz etal., U.S. Pat. No. 5,498,530; Huse et al. Science 246:1275-1281, 1989;and Lam et al., Nature 354:82-84, 1991).

The present invention also provides IL-2 mutants characterized bysubstantially reduced vasopermeability activity and substantiallysimilar binding affinity for an IL-2 receptor compared to a wildtypeform of the IL-2 mutant. Such IL-2 mutants comprise at least onemutation in the permeability enhancing peptide region of the IL-2molecule, the mutation preferably involving substitution of one or morewildtype amino acid residue in that region. Designation of various formsof IL-2 herein is made with respect to the sequence shown and numberedas in FIG. 9, noting only modifications thereof at the subscriptedpositions. Various designations may be used herein to indicate the samemutation. For example, a mutation from arginine at position 38 totryptophan can be indicated as W₃₈, W38, 38W or R38W.

IL-2 mutants with decreased vasopermeability may be mutated bysubstitution at amino acid 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, or 55 or combinations thereof. In a more preferredembodiment, the IL-2 mutant has a mutation at amino acid 38, 39, 42, or55, wherein said non-wildtype residue at position 38 is not alanine orglutamine while said non-wildtype residue at position 42 is not lysine.In an even more preferred embodiment, the IL-2 mutant is W₃₈, G₃₈, Y₃₈,L₃₉, K₄₂ and Y₅₅. These mutants exhibit substantially similar bindingaffinity to low-affinity and intermediate-affinity IL-2 receptors andhave substantially reduced vasopermeability activity as compared to awildtype form of the IL-2 mutant.

Preferable mutations may actually display increased binding affinity forthe low- and intermediate-affinity IL-2 receptors. Other characteristicsof useful mutants may include the ability to induce proliferation ofIL-2 receptor bearing T cells, a reduced ability to induce elaborationof secondary cytokines by peripheral blood mononuclear cells,particularly IL-1β and TNF-α, and a reduced toxicity profile in vivo.Mutants 38G and 55Y, which exhibit substantially reducedvasopermeability activity, but which substantially retain the ability togenerate IFN-γ as a secondary cytokine also represent IL-2 mutants ofthe invention. A particularly preferred IL-2 mutant polypeptide is 38W,which exhibits substantially reduced vasopermeability, retainssubstantial affinity for the low- and intermediate-affinity IL-2receptor, and retains 50% or more of the IL-2 dependent cell line HT-2proliferative activity of native IL-2 (Table 3).

IL-2 mutants of the invention, in addition to having a mutation in thevasopermeability region of IL-2, also may have one or more mutations inthe amino acid sequence outside this region. Mutations in human IL-2affecting position 1-21 and 59-133 can provide additional advantagessuch as increased expression or stability. For example, the cysteine atposition 125 may be replaced with a neutral amino acid such as serine,alanine, threonine or valine, yielding S₁₂₅IL-2, A₁₂₅IL-2, T₁₂₅IL-2 orV₁₂₅IL-2 respectively, as described in U.S. Pat. No. 4,518,584 (RE33,653). As described therein, one may also delete the N-terminalalanine residue of IL-2 yielding such mutants as des-A₁S₁₂₅ ordes-A₁A₁₂₅. A cysteine residue may be substituted for any non-cysteineresidue at positions 1-20 and particularly at position 3 as described inU.S. Pat. No. 5,206,344. Alternatively or conjunctively, the IL-2 mutantinclude mutation whereby methionine normally occurring at position 104of wild-type IL-2 is replaced by a neutral amino acid such as alanine(see U.S. Pat. No. 5,206,344). The resulting mutants, e.g., des-A₁A₁₀₄IL-2, des-A₁A₁₀₄S₁₂₅ IL-2, A₁₀₄IL-2, A₁₀₄A₁₂₅IL-2, des-A₁A₁₀₄A₁₂₅IL-2,or A₁₀₄S₁₂₅IL-2 may be used to conjunction with the preferred IL-2mutations of the invention that substantially reduced vasopermeabilityactivity while retaining substantially similar binding affinity for anIL-2 receptor compared to a wildtype form of the IL-2 mutant. Also, athreonine at position 3 of the native molecule can be replaced bycysteine to yield e.g., des-A₁C₃A₁₀₄IL-2, des-A₁C₃A₁₀₄S₁₂₅IL-2,C₃A₁₀₄IL-2, C₃A₁₀₄A₁₂₅IL-2, des-A₁C₃A₁₀₄ A₁₂₅IL-2, or C₃A₁₀₄S₁₂₅IL-2,each of which may be used to conjunction with the preferred IL-2mutations of the invention. In these mutants substitution removes theglycosylation site at position 3 without eliminating biological activity(see Japanese Patent Application No. 235,638 filed Dec. 13, 1983). Theseand other mutants may be found in U.S. Pat. No. 5,116,943 (see claim 5)and in Weiger et al., Eur. J. Biochem., 180:295-300 (1989).

Mutations of the invention that substantially reduce vasopermeabilityactivity while retaining substantially similar binding affinity for anIL-2 receptor compared to a wildtype form of the IL-2 mutant also may becombined with other toxicity reducing mutations such as when asparagineat position 88 is replaced by arginine (i.e., R₈₈IL-2, also known as BAY50-4798), described by Shanafelt et al., Nature Biotech. 18:1197-1202(2000). As shown in the Examples, the N88R mutant has reduced toxicitybut this does not occur by reduced vasopermeability. According toShanafelt et al., reduced toxicity for this mutant results fromdecreased binding to the intermediate affinity (NK) IL-2 receptor. Thus,an IL-2 mutant that contains both a vasopermeability reducing mutationin the vasopermeability enhancing peptide region of IL-2 as well as theN88R mutation that reduces toxicity by reducing binding to theintermediate IL-2 receptor will provide an IL-2 mutant with unique anduseful therapeutic efficacy.

IL-2 mutants of the invention can be prepared by deletion, substitution,insertion or modification using genetic or chemical methods well knownin the art. Genetic methods may include site-directed mutagenesis, PCR,gene synthesis, and the like. In this regard, the nucleotide sequence ofnative IL-2 has been described by Taniguchi et al. (Nature 302:305,1983) and nucleic acid encoding human IL-2 is available from publicdepositories such as the American Type Culture Collection (RockvilleMd.). Substitution or insertion may involve natural as well asnon-natural amino acid residues. Amino acid modification includes wellknown methods of chemical modification such as the addition ofglycosylation sites or carbohydrate attachments, and the like.

Mutant IL-2 may be prepared by recombinant expression methods such as inbacteria and yeast as described previously (see U.S. Pat. No.5,116,943). In general, nucleic acid encoding the mutant IL-2 can becloned into an expression vector for high yield expression of theencoded product. The expression vector can be part of a plasmid, virus,or may be a nucleic acid fragment. The expression vector includes anexpression cassette into which the nucleic acid encoding the IL-2 mutantis cloned in operable association with a promoter. The expressioncassette may also include other features such as an origin ofreplication, and/or chromosome integration elements such as retroviralLTRs, or adeno associated viral (AAV) ITRs. If secretion of the IL-2mutant is desired, DNA encoding a signal sequence may be placed upstreamof the nucleic acid encoding the mature amino acids of the mutant IL-2.DNA encoding a short protein sequence that could be used to facilitatelater purification (e.g., a histidine tag) or assist in labeling theIL-2 mutant may be included within or at the ends of the IL-2 mutantencoding nucleic acid. The expression vector pEE12/chTNT-3 HC/huIL-2(mutant or native) described in the Examples and which encodes a fusionprotein comprising human IL-2 (mutant or native) coupled to thecarboxy-terminus of chTNT-3 heavy chain via a non-cleavable seven aminoacid linker is one example of a useful expression vector.

Cells suitable for replicating and for supporting expression of IL-2mutants are well known in the art. Such cells may be transfected ortransduced as appropriate with the particular expression vector andlarge quantities of vector containing cells can be grown for seedinglarge scale fermenters to obtain sufficient quantities of the IL-2mutant for clinical applications. Such cells may include prokaryoticmicroorganisms, such as E. coli, or various other eukaryotic cells, suchas Chinese hamster ovary cells (CHO), insect cells, or the like.Standard technologies are known in the art to express foreign genes inthese systems. For example, the NSO marine myeloma cell line, which wastransfected with expression vector pEE12/chTNT-3 HC/huIL-2 (mutant ornative) as described in the Examples, is Suitable for supportingexpression of an antibody mutant IL-2 fusion protein.

An IL-2 mutant can be prepared where the IL-2 polypeptide segment islinked to one or more molecules such as a polypeptide, protein,carbohydrate, lipid, nucleic acid, polynucleotide or molecules that arecombinations of these molecules (e.g., glycoproteins, glycolipids etc).The IL-2 mutant also may be linked to organic moiety, inorganic moietyor pharmaceutical drug. As used herein, a pharmaceutical drug is anorganic containing compound of about 5,000 daltons or less.

The IL-2 mutant may also be linked to multiple molecules of the sametype or to more than one type of molecule. In some cases, the moleculethat is linked to IL-2 can confer the ability to target the IL-2 tospecific tissues or cells in an animal. In this embodiment, the othermolecule may have affinity for a ligand or receptor in the target tissueor cell, thereby directing the IL-2 to the target tissue or cell.Targeting molecules include, for example, antibodies specific for cellsurface or intracellular proteins, ligands of biological receptors, andthe like. Such antibodies may be specific for well known tumorassociated antigens such as carcinoembryonic antigen, the TAG-72antigen, the EGF receptor, and the like. Antibodies to DNA such as theTNT antibody described in the Examples is an example of a usefultargeting molecule that can be fused or conjugated to mutant IL-2.

The IL-2 mutant also may be linked to any biological agent includingtherapeutic compounds such as anti-neoplastic agents include paclitaxel,daunorubicin, doxorubicin, caminomycin, 4′-epiadriamycin,4-demethoxy-daunomycin, 11-deoxydaunorubicin, 13-deoxydaunorubicin,adriamycin-14-benzoate, adriamycin-14-octanoate,adriamycin-14-naphthaleneacetate, vinblastine, vincristine, mitomycin C,N-methyl mitomycin C, bleomycin A₂, dideazatetrahydrofolic acid,aminopterin, methotrexate, cholchicine and cisplatin, and the like.Anti-microbial agents include aminoglycosides including gentamicin,antiviral compounds such as rifampicin, 3′-azido-3′-deoxythymidine (AZT)and acylovir, antifungal agents such as azoles including fluconazole,plyre macrolides such as amphotericin B, and candicidin, anti-parasiticcompounds such as antimonials, and the like. Hormones may include toxinsuch as diphtheria toxin, cytokine such as CSF, GSF, GMCSF, TNF,erythropoietin, immunomodulators or cytokines such as the interferons orinterleukins, a neuropeptide, reproductive hormone such as HGH, FSH, orLH, thyroid hormone, neurotransmitters such as acetylcholine, hormonereceptors such as the estrogen receptor. Also included are non-steroidalanti-inflammatories such as indomethacin, salicylic acid acetate,ibuprofen, sulindac, piroxicam, and naproxen, and anesthetics oranalgesics. Also included are radioisotopes such as those useful forimaging as well as for therapy.

An IL-2 mutant which is a fusion between IL-2 and another polypeptidecan be designed such that the IL-2 sequence is fused directly to thepolypeptide or indirectly through a linker sequence. The composition andlength of the linker may be determined in accordance with methods wellknown in the art and may be tested for efficacy. An example of a linkersequence between IL-2 and an antibody heavy chain is shown in FIG. 2.Additional sequences may also be included to incorporate a cleavage siteto separate the individual components of the fusion if desired, forexample an endopeptidase recognition sequence. In addition, an IL-2mutant may also be synthesized chemically using methods of polypeptidesynthesis as is well known in the art (e.g., Merrifield solid phasesynthesis).

As used herein, “antibody” is intended to include all forms of anantibody, including all natural and unnatural antibody forms. Thisincludes the typical antibody that consists of four subunits includingtwo heavy chains and two light chains, domain-deleted antibodies, Fabfragments, Fab′2 fragments, Fv fragments, single chain Fv antibodies,and the like. An antibody also includes the heavy chain alone or thelight chain alone. Methods to produce polyclonal antibodies andmonoclonal antibodies are well known in the art (see, e.g., Harlow andLane, “Antibodies, a laboratory manual.” Cold Spring Harbor Laboratory,1988). Non-naturally occurring antibodies can be constructed using solidphase peptide synthesis, can be produced recombinantly or can beobtained, for example, by screening combinatorial libraries comprisingvariable heavy chains and variable light chains (see, e.g., U.S. Pat.No. 5,969,108 to McCafferty).

IL-2 may be genetically fused to single polypeptide antibody forms ormay be chemically conjugated to any of the antibody forms. Fusion ofIL-2 to an antibody heavy chain is described in the Examples. Any animalspecies of antibody can be linked to a mutant IL-2. If the mutantIL-2/antibody conjugate or fusion is intended for human use, a chimericform of the antibody may be used wherein the constant regions of theantibody are from a human. A fully humanized form of the antibody canalso be prepared in accordance with methods well known in the art (see,e.g., U.S. Pat. No. 5,565,332 to Winter). Cells expressing a mutant-IL-2fused to either the heavy or the light antibody chain may be engineeredso as to also express the other of the antibody chains such that theexpressed mutant IL-2 fusion product is an antibody that has both aheavy and a light chain.

Mutant IL-2 may be chemically conjugated to another molecule using wellknown chemical conjugation methods. Bi-functional cross-linking reagentssuch as homofunctional and heterofunctional cross-linking reagents wellknown in the art can be used for this purpose. The type of cross-linkingreagent to use depends on the nature of the molecule to be coupled toIL-2 and can readily be identified by those skilled in the art.Alternatively, or in addition, mutant IL-2 and/or the molecule to whichit is intended to be conjugated may be chemically derivatized such thatthe two can be conjugated in a separate reaction as is also well knownin the art.

IL-2 mutants prepared as described herein may be purified by biochemicalmethods well known in the art. Such methods may include affinitychromatography such as binding and elution to a ligand or antigen towhich the fusion protein is reactive. For example, sequential Protein Aaffinity chromatography, and ion-exchange chromatography can be used toisolate a fusion protein (or conjugate) essentially as described in theExamples. The purity of the mutant IL-2 fusion protein can be determinedby any of a variety of well known analytical methods including gelelectrophoresis, high pressure liquid chromatography, and the like. Forexample, the chimeric heavy chain fusion proteins expressed as describedin the Examples were shown to be intact and properly assembled asdemonstrated by reducing SDS-PAGE (FIG. 3). Two bands were resolved forchTNT-3/huIL-2 at approximately M_(r) 25,000 and M_(r) 70,000,corresponding to the predicted molecular weights of the immunoglobulinlight chain and heavy chain/IL-2 fusion protein.

Further chemical modification of the IL-2 mutant polypeptide may bedesirable. For example, problems of immunogenicity and short half-lifemay be improved by conjugation to substantially straight chain polymerssuch as polyethylene glycol (PEG) or polypropylene glycol (PPG) (see,e.g., PCT WO87/00056).

In accordance with another aspect of the present invention, there isprovided a method for stimulating the immune system of an animal byadministering the IL-2 mutants of the invention. The method is useful totreat disease states where the host immune response is deficient. Intreating a subject, a therapeutically effective dose of compound (i.e.,active ingredient) is administered. A therapeutically effective doserefers to that amount of the active ingredient that producesamelioration of symptoms or a prolongation of survival of a subject. Aneffective dose will vary with the characteristics of the IL-2 mutant tobe administered, the physical characteristics of the subject to betreated, the nature of the disease or condition, and the like. A singleadministration can range from about 50,000 IU/kg to about 1,000,000IU/kg or more, more typically about 600,000 IU/kg. This may be repeatedseveral times a day (e.g., 2-3×), for several days (e.g., about 3-5consecutive days) and then may be repeated one or more times following aperiod of rest (e.g., about 7-14 days). Thus, an effective dose maycomprise only a single administration or many administrations over aperiod of time (e.g., about 20-30 individual administrations of about600,000 IU/kg each given over about a 10-20 day period).

Disease states for which the mutant IL-2 can be administered comprise,for example, a tumor or infection where a cellular immune response wouldbe a critical mechanism for specific immunity. Stimulation of the immunesystem may include any one or more of a general increase in immunefunction, an increase in T cell function, a restoration of lymphocytefunction, an increase in the expression of IL-2 receptors, an increasein T cell responsiveness, an increase in natural killer cell activity orlymphokine-activated killer cell activity, and the like. Illustrative ofspecific disease states for which IL-2 mutants of the present inventioncan be employed include cancer, specifically renal cell carcinoma ormelanoma; immune deficiency, specifically in HIV-positive patients,immunosuppressed patients, and autoimmune disorders, chronic infectionand the like.

The IL-2 mutant may be administered in combination with one or moretherapeutic agents, for example, a cytokine, antiviral or antifungalagent. The term “therapeutic agent” encompasses any agent administeredto treat a symptom or disease in an animal in need of such treatment.The IL-2 mutant may also be administered as a component of a vaccine,i.e. combined with essentially any preparation intended for activeimmunological prophylaxis.

Toxicity and therapeutic efficacy of an IL-2 mutant can be determined bystandard pharmaceutical procedures in cell culture or experimentalanimals (see, e.g. Example 3B). Cell culture assays and animal studiescan be used to determine the LD₅₀ (the dose lethal to 50% of apopulation) and the ED₅₀ (the dose therapeutically effective in 50% of apopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index, which can be expressed as the ratio LD₅₀/ED₅₀. IL-2mutants that exhibit large therapeutic indices are preferred. The dataobtained from these cell culture assays and animal studies can be usedin formulating a range of dosages suitable for use in humans. The dosageof such mutants lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon a variety of factors,e.g., the dosage form employed, the route of administration utilized,the condition of the subject, and the like.

A therapeutically effective dose can be estimated initially from cellculture assays by determining an IC₅₀. A dose can then be formulated inanimal models to achieve a circulating plasma concentration range thatincludes the IC₅₀ as determined in cell culture. Such information can beused to more accurately determine useful doses in humans. Levels inplasma may be measured, for example, by HPLC. The exact formulation,route of administration and dosage can be chosen by the individualphysician in view of the patient's condition.

The attending physician for patients treated with IL-2 mutants wouldknow how and when to terminate, interrupt, or adjust administration dueto toxicity, organ dysfunction, and the like. Conversely, the attendingphysician would also know to adjust treatment to higher levels if theclinical response were not adequate (precluding toxicity). The magnitudeof an administered close in the management of the disorder of interestwill vary with the severity of the condition to be treated, with theroute of administration, and the like. The severity of the conditionmay, for example, be evaluated, in part, by standard prognosticevaluation methods. Further, the dose and perhaps dose frequency willalso vary according to the age, body weight, and response of theindividual patient.

IL-2 mutants of the invention may be administered to an individual aloneas a pharmaceutical preparation appropriately formulated for the routeof delivery and for the condition being treated. Suitable routes mayinclude oral, rectal, transdermal, vaginal, transmucosal, or intestinaladministration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections, and the like. For transmucosal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

IL-2 mutants may be manufactured as a formulation with one or morepharmaceutically acceptable carriers or excipient(s) as is well known inthe art. Techniques for formulation and administration may be found in“Remington's Pharmaceutical Sciences,” (18th ed., Mack Publishing Co.,Easton, Pa., 1990). Specific examples of IL-2 formulations are describedin U.S. Pat. Nos. 4,604,377 and 4,766,106. The IL-2 mutant may beformulated as a liquid with carriers that may include a buffer and orsalt such as phosphate buffered saline. Alternatively, the IL-2 mutantmay be formulated as a solid with carriers or fillers such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers.

For oral delivery, the formulated end product may be a tablet, pill,capsule, dragee, liquid, gel, syrup, slurry, suspension, and the like.Also, push-fit capsules made of gelatin, as well as soft, sealedcapsules made of gelatin and a plasticizer, such as glycerol or sorbitolmay be used. The push-fit capsules can contain the active ingredients inadmixture with fillers as above while in soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid paraffin, or liquid polyethylene glycols.

Formulation for oral delivery may involve conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, lyophilizing processes, and the like. TheIL-2 mutant also may be mixed with a solid excipient, optionallygrinding the resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are, in particular, fillers such assugars, including lactose, sucrose, mannitol, sorbitol, and the like;cellulose preparations such as, for example, maize starch, wheat starch,rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose,polyvinylpyrrolidone (PVP), and the like, as well as mixtures of any twoor more thereof. If desired, disintegrating agents may be added, such ascross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereofsuch as sodium alginate, and the like.

If injection is desired, the IL-2 mutant may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHank's solution, Ringer's solution, or physiological saline buffer.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain compounds which increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol,dextran, or the like. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

The present invention also provides a method of producing a low toxicityIL-2 in a form suitable for administration in vivo, said methodcomprising:

a) Obtaining a mutant IL-2 characterized by substantially reducedvasopermeability activity and substantially similar binding affinity foran IL-2 receptor compared to a wildtype form of the IL-2 mutant; and

b) formulating the mutant IL-2 with at least one pharmaceuticallyacceptable carrier, whereby a preparation of low toxicity IL-2 isformulated for administration in vivo. In this aspect, the mutant IL-2may be obtained by culturing a recombinant organism containing nucleicacid encoding the mutant IL-2 or by producing the mutant IL-2 by invitro chemical synthesis.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLES Example 1 Reagents

This example provides the preferred reagents for practice of theembodied invention. One skilled in the art can readily appreciatecomparable materials that can be substituted in place of these reagents.

The Glutamine Synthase Gene Amplification System, including theexpression plasmids pEE6/hCMV-B and pEE12 as well as the NS0 murinemyeloma expression cell line, were purchased from Lonza Biologics(Slough, UK). Restriction endonucleases, T4 DNA ligase, Vent polymerase,and other molecular biology reagents were purchased from either NewEngland Biolabs (Beverly, Mass.) or Boehringer Mannheim (Indianapolis,Ind.). Dialysed fetal bovine serum, crude DNA from salmon testes,single-stranded DNA from calf thymus, chloramine T, and2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt(ABTS) were purchased from Sigma Chemical Co. (St. Louis, Mo.).Recombinant human interleukin-2 was purchased from Chiron (Emeryville,Calif.). The Griess Reagent System, containing sulfanilamide solution,N-1-naphthylethylenediamine dihydrochloride solution, and nitritestandards, was purchased from the Promega Corporation (Madison, Wis.).¹²⁵I was obtained from DuPont New England Nuclear (North Billerica,Mass.) as sodium iodide in 0.1 N sodium hydroxide. BALB/c mice wereobtained from Harlan Sprague-Dawley (Indianapolis, Ind.).Sulibsuccinimidyl 6-(biotinamido) hexanoate (Sulfo-NHS-LC biotin) waspurchased from Pierce (Rockford, Ill.). HRPO-conjugated secondaryreagents (goat-anti-human IgG (FcSp) and streptavidin) were purchasedfrom CalTag (Burlingame, Calif.).

The Daudi lymphoma cell line (Ohsugi et al., J. Nat. Cancer Inst.65:715. 1980), HT-2 lymphoma line (Shipley et al., Cell. Immunol.93:459, 1985), and LS 174T human colorectal carcinoma cell line (Tom etal., In Vitro 112:180, 1976) were obtained from the American TypeCulture Collection (Manassas, Va.). The Madison 109 murine lungadenocarcinoma (Marks et al., Cancer Treatment Reports 61:1459, 1977)was obtained from the National Cancer Institute (Frederick, Md.). TheMT-1 human T lymphotropic virus-1-transformed T cell line (Tsudi et al.,J. Immunol. 143:4039, 1989) and YT-2C2 cell line, a subclone of theacute lymphoblastic lymphoma cell line YT (Yodoi et al., J. Immunol.134:1623, 1985), were generous gifts of Thomas L. Ciardelli (DartmouthMedical School).

Example 2 Development and Characterization of IL-2 Mutant Polypeptides

This example provides methods of creating IL-2 mutant polypeptides andchimeric antibody/IL-2 fusion proteins (mutant or native). In addition,this example provides methods for determining the cytokine function andbinding properties of resultant IL-2 molecules in vitro.

A. Construction and Expression of IL-2 and Antibody/IL-2 Fusion Proteins

The construction of the chimeric monoclonal antibody TNT-3 (chTNT-3,IgG₁ κ) and the fusion protein of this antibody with IL-2 have beenpreviously described (Hornick et al., Cancer Biotherapy &Radiopharmaceuticals 13:255, 1998; Hornick et al., J. Nucl. Med 41:355,2000).

IL-2 mutant cDNA was prepared by site-directed mutagenesis to mutateamino acid 20 from aspartic acid to lysine (D20K), amino acid 38 fromarginine to glycine (R38G) or tryptophan (R38W), amino acid 39 frommethionine to valine (M39V) or leucine (M39L), amino acid 42 fromphenylalanine to lysine (F42K), and amino acid 55 from histidine totyrosine (H55Y) using the following 5′ and 3′ primer pairs,respectively:

(SEQ ID NO. 4) D20K-5′-TTACTGCTGA AATTACAGA TG-3′, and (SEQ ID NO. 5)5′-CATCTGTAAT TTCAGCAGTA A-3′; (SEQ ID NO. 6)R38G/W-5′-AAACTCACC(G/T) GGATGCTCAC A-3′, and (SEQ ID NO. 7)5′-TGTGAGCATC C(A/C)GGTGAGTT T-3′; (SEQ ID NO. 8)M39V/L-5′-CTCACCAGG(G/C) TGCTCACATT T-3′, and (SEQ ID NO. 9)5′-AAATGTGAGC A(G/C)CCTGGTGA G-3′; (SEQ ID NO. 10)F42K-5′-ATGCTCACAA AGAAGTTTTA C-3′, and (SEQ ID NO. 11)5′-GTAAAACTTC TTTGTGAGCA T-3′; and (SEQ ID NO. 12)H55Y-5′-GAACTGAAAT AATCTTCAGT GT-3′, and (SEQ ID NO. 13)5′-ACACTGAAGA TATTTCAGTT C-3′.

IL-2 mutant cDNA was similarly prepared to mutate amino acid 38 fromarginine to tyrosine (R38Y) or to glutamic acid (R38E).

The full-length IL-2 mutant was then amplified by PCR with the followingprimers:

(SEQ ID NO. 14) 5′-GGTAAAGCGG CCGCAGGAGG TGGTAGCGCA CCTACTTCAAGTTCTACA-3′; and (SEQ ID NO. 15)5′-TCATGCGGCC GCTCAAGTTA GTGTTGAGAT GATGCT-3′,

which appended a NotI restriction site and codons for a polypeptidelinker to the 5′ end, and a stop codon and NotI site at the 3′ end ofthe IL-2 mutant cDNA.

The resulting PCR product was then restricted with Not I and cloned intothe Not I restricted pEE12/chTNT-3 HC expression vector to produce thechTNT-3/IL-2 mutant fusion construct (see FIG. 2). Constructs wereintroduced in to target cells using standard electroporation techniques.These fusion proteins were expressed from NS0 murine myeloma cells forlong term stable expression according to the manufacturer's protocol(Lonza Biologics). The highest producing clone was scaled up forincubation in a 3 L stir flask bioreactor and the fusion proteinpurified from the spent culture medium by sequential Protein A affinitychromatography and ion-exchange chromatography, using methods known inthe art. The fusion protein was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditionsand stained with Coomassie blue to demonstrate proper assembly andpurity (see FIG. 3).

chTNT-3/IL-2 mutant-secreting clones were initially identified byindirect ELISA analysis of supernatants using microtiter plates coatedwith crude DNA preparations from calf thymus at 50 μg/mL to detectbinding of the TNT antibody portion of the fusion protein. Followingthis initial screening, production rate assays were performed byincubating 1×10⁶ cells in 1 mL of selective medium for 24 hours, afterwhich the supernatants were analyzed by indirect ELISA analysis usingmicrotiter plates coated with single-stranded DNA preparations fromsalmon testes at 100 μg/mL. Detection of chTNT-3 and chTNT-3 fusionproteins bound to the DNA antigen was accomplished withhorse-radish-peroxidase-conjugated goat-anti-human IgG (FcSp) followedby color development produced by enzymatic cleavage of ABTS. Dilutionsof chTNT-3 were used to generate a standard curve using a 4-parameterfit by an automated ELISA reader (Bio-Tek Instruments, Winooski, Vt.),from which concentrations of unknowns were estimated and expressed asμg/mL/10⁶ cells/24 hours.

B. Determination of IL-2 Receptor Binding

The purified antibody/IL-2 fusion proteins were examined for theirability to bind to different forms of the IL-2 receptor using variousavailable cell lines. Table 1 shows the characteristics of IL-2receptors and expressing cell lines.

TABLE 1 Interleukin-2 Receptors and Native IL-2 Binding AffinityReceptor Protein Affinity Cell Line Low-Affinity α (p55) K_(d) = 10⁻⁸ MMT-1 Intermediate- βγ (p70, p75 complex) K_(d) = 10⁻⁹ M YT-2C2 AffinityHigh-Affinity α βγ (p55 and p70, p75 K_(d) = 10⁻¹¹ M HT-2 complex)

Relative binding studies were performed on MT-1 and YT-2C2 cell linesusing the method of Frankel and Gerhard (Mol. Immunol. 16:101, 1979) todetermine the avidity constant of the antibody/IL-2 mutant fusionproteins to the low- and intermediate-affinity IL-2 receptors,respectively. The MT-1 cell line is an HTLV-I-transformed T cell linethat lacks IL-2Rβ expression (i.e., only expresses IL-2Rα and γ) (Oda etal., Intl. Immunol. 9:1303, 1997). In contrast, the YT-2C2 cell line, asubclone of the acute lymphoblastic lymphoma YT cell line, is an NK-likecell line that lacks IL-2Rα expression and thus only expresses IL-2Rβand γ (Yodoi et al., J. Immunol. 134:1623, 1985; Farrier et al., Blood8:4568, 1995).

Cells were harvested and dead cells were removed by Ficoll-Hypaquedensity centrifugation to remove cells with exposed DNA that could bindto the TNT-3 portion of the antibody/IL-2 fusion protein. The purifiedviable cells were then used in IL-2 binding studies within one hour ofpurification. These target cells were incubated with 10 to 100 ng of¹²⁵I-labeled chTNT-3/IL-2 fusion protein or mutant fusion protein in PBSfor 30 minutes at room temperature with constant mixing. This shortincubation period was chosen to allow sufficient time for the bindingand internalization of the IL-2 containing proteins, but insufficienttime for the cell to metabolize these proteins. To minimize contributionof the antibody moiety to fusion protein binding to the target cells, a10-fold molar excess of unlabelled antibody was used to prevent bindingof the TNT-3 portion of the fusion protein to the cells. The activity inthe supernatants after cell removal was then measured in a gamma counterand the amount of bound radioactivity (cpm) determined by subtractiveanalysis. The amount of bound fusion protein was then calculated fromthe cell-bound radioactivity and the specific activity (cpm/ng) of theradiolabeled antibody preparation. Scatchard plot analysis was used toobtain the slope. The equilibrium or avidity constant K_(a) wascalculated by the equation K_(a)=−(slope/n), where n is the valence ofthe fusion protein (2 for IgG fusion protein).

TABLE 2 IL-2 Receptor Binding Affinity of chTNT-3/IL-2 and chTNT-3/IL-2Mutant Fusion Proteins ChTNT-3 Antibody/IL-2 *Low-affinity IL-2#Intermediate-affinity Fusion Protein Receptor IL-2 Receptor IL-2 Native1.18 × 10⁹ 1.18 × 10⁹ D20K IL-2 Mutant 1.61 × 10⁹ 0.57 × 10⁹ R38G IL-2Mutant 1.35 × 10⁹ 1.56 × 10⁹ R38W IL-2 Mutant 1.20 × 10⁹ 1.63 × 10⁹ M39VIL-2 Mutant 1.18 × 10⁹ 1.37 × 10⁹ M39L IL-2 Mutant 1.02 × 10⁹ 1.43 × 10⁹F42K IL-2 Mutant 1.50 × 10⁹ 0.90 × 10⁹ H55Y IL-2 Mutant 0.90 × 10⁹ 1.34× 10⁹ *Performed using MT-1 cells. #Performed using YT-2C2 cells.

The results of IL-2 receptor binding to the various antibody/IL-2 fusionproteins shown in Table 2 indicate that the majority of antibody/IL-2mutant fusion proteins demonstrated similar binding profiles with minorvariability compared to the native fusion protein. The R38W mutantIL-2/antibody fusion protein displayed increased affinity for both thelow- and intermediate-affinity IL-2 receptors. The D20K and F42K mutantIL-2/antibody fusion proteins displayed decreased affinity for theintermediate-affinity IL-2 receptor and an increased affinity to thelow-affinity IL-2 receptor relative to the native fusion protein. Incontrast, the H55Y mutant IL-2/antibody fusion protein showed reducedaffinity to the low-affinity IL-2 receptor with minimal alteration inintermediate-affinity IL-2 receptor binding.

C. Determination of IL-2 Proliferation Activity

The purified antibody/IL-2 fusion proteins were examined for theirability to stimulate proliferation in cell-based assays utilizing themurine IL-2-dependent cell line HT-2 (Buttke et al., J. Immunol. Meth.157:233, 1993; Gieni et al., J. Immunol. Meth. 187:85, 1995). Briefly,freshly harvested HT-2 cells were washed three times with sterile PBS toremove residual IL-2. The cells were placed in sterile 96-wellflat-bottomed tissue culture plates in duplicate at 1×10⁵ cells/mL withcomplete RPMI medium or RPMI medium supplemented with a recombinant IL-2standard (rhu IL-2), chTNT-3, chTNT-3/IL-2 fusion protein orchTNT-3/IL-2 mutant fusion protein, and incubated in a 5% CO₂, 37° C.humidified atmosphere. After 72 hours, relative IL-2-dependent cellularproliferation was determined utilizing the CellTiter 96® AQueous OneSolution Cell Proliferation Assay (Promega, Madison, Wis.), a one-stepcolorimetric method that determines the relative conversion of thetetrazolium compound MTS to a colored formazan product. The absorbanceof each sample at 490 nm was determined using a Bio-Tek plate reader andthe results were graphed to determine the specific activities (IU/mg) ofthe fusion proteins.

TABLE 3 Relative ability of chTNT-3/IL-2 and chTNT-3/IL-2 mutant fusionproteins stimulate the IL-2 dependent HT-2 cell line. ChTNT-3Antibody/IL-2 IL-2 Proliferation Fusion Protein Activity (HT-2) ChTNT-3− ChTNT-3/IL-2 Native ++++ ChTNT-3/D20K IL-2 Mutant − ChTNT-3/38G IL-2Mutant + ChTNT-3/R38W IL-2 Mutant +++ ChTNT-3/R38Y IL-2 Mutant ++ChTNT-3/R38E IL-2 Mutant − ChTNT-3/M39V IL-2 Mutant + ChTNT-3/M39L IL-2Mutant + ChTNT-3/F42K IL-2 Mutant + ChTNT-3/H55Y IL-2 Mutant + Expressedas percent of native IL-2 activity: − = no activity, + = less than 25%activity, ++ = 25-50% activity, +++ = 51-75% activity, ++++ = 76-100%.

The results presented in Table 3 show that the majority of theantibody/IL-2 mutant fusion proteins retained their ability to stimulateproliferation of HT-2 cells, with the exception of the D20K and R38Emutant IL-2/antibody fusion proteins. Notably, the R38W mutantIL-2/antibody fusion protein exhibited 51-75% activity in comparison tothe native IL-2/antibody fusion protein. It also is noted that the N88RIL-2 mutant showed strong IL-2 proliferative activity, similar inmagnitude to that seen for the R38W IL-2 mutant.

D. Quantitation of Secondary Cytokine Induction

The purified antibody/IL-2 fusion proteins were examined for theirability to induce the expression of the cytokines interleukin-1β(IL-1β), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) fromhuman peripheral blood mononuclear cells (PBMC) using indirect ELISAanalysis. Freshly purified human PBMC were isolated from healthy normaldonors by leukopheresis and fractionated on Histopaque 1077(Sigma-Aldrich, St. Louis, Mo.) by centrifugation at 450 g for 30minutes. Cells were stimulated with 1 nM chTNT-3, chTNT-3/IL-2 fusionprotein, or chTNT-3/IL-2 mutant fusion protein at 1×10⁶ cells/mL in a 5%CO₂ humidified 37° C. incubator. AIM-V serum-free lymphocyte media (LifeTechnologies, Rockville, Md.) was utilized to eliminate the effect ofserum on cytokine induction. Supernatants were collected after one,three, five, and seven days, centrifuged to remove remaining cells, andcytokine concentrations determined by ELISA following the manufacturer'sprotocol (Endogen, Inc., Woburn, Mass.). Absorbance, was detected byspectrophotometry, and the concentration of cytokine was determined froma standard curve. The mean cytokine secretion was determined bystandardizing the mutant-stimulating cytokine secretion as a percentageof the mean rhuIL-2-induced secretion for each day in each individualexperiment. The sensitivity of each ELISA varied from 3-10 pg/mL. Theresults are summarized in Table 4 and in FIGS. 4A-4C.

TABLE 4 Relative ability of chTNT-3/IL-2 and chTNT-3/IL-2 mutant fusionproteins to induce secondary cytokine production. Secondary CytokineProduction IL-1β IFN-γ TNF-α ChTNT-3 − − − ChTNT-3/Native IL-2 ++++ ++++++++ ChTNT-3/D20K IL-2 Mutant −/+ −/+ −/+ ChTNT-3/R38G IL-2 Mutant ++ ++++ ChTNT-3/R38W IL-2 Mutant ++ ++++ ++ ChTNT-3/M39V IL-2 Mutant +++ ++++++ ChTNT-3/M39L IL-2 Mutant ++ +++ ++ ChTNT-3/F42K IL-2 Mutant −/+ + −ChTNT-3/H55Y IL-2 Mutant ++ ++++ ++ Expressed as percent of nativeactivity: − = no activity, + = less than 25% activity, ++ = 25-50%activity, +++ = 51-75% activity, ++++ = 76-100%.

The results show that the D20K and F42K mutant IL-2/antibody fusionproteins were unable to elicit the production of the cytokines IL-1β,IFN-γ, and TNF-α, while the R38G, R38W, M39V, M39L, H55Y and N88R mutantIL-2/antibody fusion proteins retained ≧50% of the activity of thenative IL-2/antibody fusion protein in inducing secondary cytokineproduction. The choice of replacement amino acid at the same positionalso effected secondary cytokine production. For example, the R38Wmutant retained 76-100% of the activity of the native IL-2 fusionprotein in inducing IFN-γ production, while the R38G mutant retainedonly 25-50% of the activity of the native IL-2 fusion protein.

E. Determination of Lymphokine-Activated Killer (LAK) Cell Activity

The purified antibody/IL-2 fusion proteins were examined for theirability to stimulate LAK cell activity. PBMC were cultured at 1×10⁶cells/mL in AIM-V medium in the presence of 1 nM chTNT-3, rhuIL-2,chTNT-3/IL-2 fusion protein, or chTNT-3/IL-2 mutant fusion protein andincubated at 37° C. in a humidified 5% CO₂ atmosphere. AIM-V (LifeTechnologies, Inc., Rockville, Md.) is a chemically defined serum-freemedia designed to support the growth of lymphocytes in the absence ofserum, thereby avoiding the serum-induced activation of PBMC. After 72hours, the cells were harvested, washed, and incubated with Daudilymphoma cells in four hour cytotoxicity assays. Lactate dehydrogenase(LDH) release was measured with the Promega CytoTox96 Non-RadioactiveCytotoxicity Assay. Spontaneous LDH release from target and effectorcells were both subtracted from the measured values and the finalresults were expressed in percent specific cytotoxicity. The resultsshown in FIGS. 5A-5C indicate that the R38G and the R38W antibody/IL-2mutant fusion proteins were capable of activating PBMC to generate LAKactivity.

Example 3 Characterization of IL-2 Mutant Polypeptide Activities In Vivo

This example provides methods of determining the in vivo activity ofchimeric antibody/IL-2 fusion proteins (mutant or native). Specifically,this example provides methods for determining the toxicity andimmunotherapeutic properties of IL-2 fusion proteins.

A. Determination of IL-2 Vasopermeability Activity

In order to determine whether the IL-2 mutant polypeptides exhibitedreduced toxicity, vasopermeability activity was monitored in vivo.Six-week old BALB/c nu/nu mice were inoculated subcutaneously in theleft flank with approximately 1×10⁷ LS174T human colorectal carcinomacells. Approximately 10 days later, when the tumors had reachedapproximately 0.5-1.0 cm in diameter, the mice were injectedintravenously with a 0.1 mL inoculum containing 25 μg of chTNT-3antibody alone, chTNT-3/native IL-2 fusion protein, or chTNT-3/IL-2mutant fusion protein (n=5/group). Two hours later, the animals wereinjected with a 0.1 mL inoculum of ¹²⁵I-B72.3, an antibody thatrecognizes TAG-72, a tumor associated glycoprotein highly expressed onhuman colorectal carcinoma. Animals were sacrificed by sodiumpentobarbital overdose three days post-injection and blood, tumor, andvarious organs were removed and weighed. The radioactivity in thesamples was then measured in a gamma counter and the data for each mousewere expressed as median percent injected dose/gram (% ID/g) and mediantumor:organ ratio (cpm per gram tumor/cpm per gram organ).Vasopermeability was expressed as the percent of thepretreatment-mediated increase in B72.3 uptake'(% ID/g) overpretreatment with chTNT-3 antibody alone. Wilcoxon rank sum analysis iswas performed to detect statistically significant differences in thebiodistribution of the molecules (p≦0.05).

TABLE 5 Vasopermeability Analysis of chTNT-3/IL-2 and chTNT-3/IL-2Mutant Fusion Proteins. Vasopermeability Induction Pretreatment (% ± sd)chTNT-3 0 ± 5 chTNT-3/IL-2 Native 100 ± 15  chTNT-3/D20K IL-2 Mutant −28± 6  chTNT-3/R38G IL-2 Mutant −7 ± 15 chTNT-3/R38W IL-2 Mutant  4 ± 16chTNT-3/R38Y IL-2 Mutant 42 ± 8  chTNT-3/R38E IL-2 Mutant −5 ± 6 chTNT-3/M39V IL-2 Mutant 99 ± 27 chTNT-3/M39L IL-2 Mutant 52 ± 23chTNT-3/F42K IL-2 Mutant 97 ± 31 chTNT-3/H55Y IL-2 Mutant −6 ± 6 

The results summarized in Table 5 show that the D20K, R38C, R38W, R38Eand H55Y antibody/IL-2 mutant fusion proteins exhibit substantiallyreduced vasopermeability activity in vivo as compared to the native IL-2antibody fusion protein. This is in contrast to the N88R mutant whichretains full vasopermeability activity.

B. Determination of Toxicity of Native and R38W Mutant IL-2 AntibodyFusion Proteins

The general comparative toxicity of the R38W mutant antibody fusionprotein as compared to the native IL-2 antibody fusion protein wasdetermined in normal 8 week-old female BALB/c mice. Mice are much lesssusceptible to IL-2 toxicity than humans. For these studies, groups of 5mice received increasing concentrations of fusion protein (10-75 μg) bydaily intravenous 0.1 mL inoculums for five consecutive days. Acutetoxicity was measured by the death of the mice.

TABLE 6 Toxicity of native and mutant IL-2 antibody fusion proteins inBALB/c mice treated intravenously for five consecutive days. FusionProtein* 10 μg 25 μg 50 μg 75 μg 100 μg ChTNT-3/IL-2 (wt) 0/5 2/5 5/55/5 5/5 ChTNT-3/R38W 0/5 0/5 0/5 2/5 5/5 ChTNT-3/N88R 0/5 0/5 0/5 0/50/5 *Data expressed as number of mice dead over total number injected.

The results in Table 6 show that the native IL-2 antibody fusion proteinwas acutely toxic in animals receiving the 25 μg dose and the higherdoses of 50 μg and 75 μg resulted in the death of all 5 mice in eachgroup. By contrast, the R38W mutant antibody fusion protein exhibiteddecreased toxicity since only 2/5 mice died at the highest dose of 75μg. These data demonstrate that the R38W mutant IL-2 shows significantlylower general toxicity than native IL-2. The N88R IL-2 mutant was evenless toxic that R38W, with all animals surviving even at a dose of 100μg. In addition, the half-life of the antibody/IL-2 fusion protein wasapproximately 12-18 hours compared to free IL-2 which has a half-life ofabout 20 minutes after intravenous administration. This shows that theIL-2 mutant antibody fusion protein is capable of prolongedadministration in vivo while remaining less toxic than native IL-2.

C. Immunotherapy of Solid Tumor with Native and R38W Mutant IL-2Antibody Fusion Proteins

In order to determine the comparative immunotherapeutic effect of theR38W mutant antibody fusion protein compared to the native IL-2 antibodyfusion protein, the proteins were administered to normal 6 week-oldfemale BALB/c mice which had been inoculated subcutaneously with 10⁷viable MAD 109 lung carcinoma cells. After 5 days, when the tumorsreached approximately 0.5 cm in diameter, groups of 5 mice receivedintravenous treatment for four consecutive days with increasing doses ofeither chTNT-3/native IL-2 or chTNT-3/R38W mutant IL-2 fusion proteinusing a 0.1 mL inoculum given once on days 5-8. Control mice received notreatment or antibody alone. Volumetric measurements of tumor size weremade three times a week starting at the time of the first therapeuticdose.

The results are shown in FIGS. 6-8. As shown in FIGS. 6A and 6B, thenative IL-2 antibody fusion protein administered to MAD 109 tumorbearing BALB/C mice showed a marked and similar decrease in tumor sizeat the all doses up through during days 5-9. Thereafter, the tumorsbegan to increase in size at roughly the same rate as untreated controlsexcept at the highest dose (50 μg).

FIG. 7A show that groups of mice receiving lower doses (5-20) of theR38W mutant IL-2/antibody fusion protein also showed similar curves asthe mice treated with the native IL-2 antibody fusion protein. Incontrast, FIG. 7B shows that mice treated with higher doses of R38W(20-50 μg) showed a slower rate of growth compared to the control miceafter discontinuation of therapy (see decreased slope in FIG. 7B versusthat of FIG. 6B).

FIG. 8 shows tumor immunotherapy for the N88R IL-2 mutant at the 5, 20and 50 μg dose. Slightly improved therapeutic affect was observed forthis mutant at the 50 μg dose as compared to native IL-2 fusion protein.Thus, these data demonstrate that significantly higher doses of the R38Wand N88R mutant IL-2 fusion protein can be used to achieve a tumorimmunotherapeutic effect that are possible with native IL-2 fusionprotein. The ability to use increased doses with reduced toxicityallowed greater tumor therapeutic effect with the low vasopermeabilityIL-2 mutants than the native IL-2.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed. The present invention may suitably bepracticed in the absence of any element or limitation not specificallydisclosed herein. The terms and expressions employed herein have beenused as terms of description to facilitate enablement and not oflimitation, and there is no intention in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof. Any cited references, to the extent thatthey provide exemplary procedural or supplementary information to thatprovided within this written description, are specifically incorporatedherein by reference.

That which is claimed is:
 1. A method of decreasing solid tumor size ina cancer patient having a cancer that is responsive to interleukin-2therapy, said method comprising administering an effective amount of aninterleukin-2 (IL-2) mutant with reduced vasopermeability activitycompared to a wildtype form of the IL-2 mutant, said mutant beingHis55Tyr, wherein said wildtype form of the human IL-2 mutant is humanIL-2 having the sequence corresponding to SEQ ID NO.
 3. 2. The method ofclaim 1, wherein said IL-2 mutant is linked to an antibody.
 3. Themethod of claim 2, wherein said antibody is a tumor targeting antibody.4. The method of claim 3, wherein said tumor targeting antibody ischTNT-3.
 5. The method of claim 1, wherein said IL-2 mutant furthercomprises a mutation outside the permeability enhancing peptide regionof IL-2.
 6. The method of claim 5, wherein said mutant further comprisesa mutation at one or more of positions 1-21 or 59-133 of IL-2.
 7. Themethod of claim 6, wherein said IL-2 mutant further comprises a mutationin a lysine at position
 88. 8. The method of claim 1, wherein saidcancer patient has renal cell carcinoma.
 9. The method of claim 1,wherein said cancer patient has melanoma.
 10. The method of claim 1,wherein said IL-2 mutant is administered in combination with atherapeutic agent.
 11. The method of claim 1, wherein said IL-2 mutantis administered as a component of a vaccine.
 12. The method of claim 1,wherein said IL-2 mutant comprises a full-length IL-2 molecule.